Ryanodine receptors (RyR) form a family of intracellular Ca2+ release channels. There is co-expression of two RyR isoforms in many vertebrate skeletal muscles and SR Ca2+ release in these muscles appears to utilize both isoforms. We do not know how a two-RyR system operates and the goal of this project is to establish the properties of this system and how it contributes to the activation of muscle contraction. In addition, we will compare Ca2+ release in these muscles with that in cardiac muscle where a single RyR isoform appears to be expressed. We will test the hypothesis that the two RyRs operate in series with each isoform being specialized for activation by interactions with either the dihydropyridine receptor (DHPR) or Ca2+. We will use skeletal muscle preparations: that express each RyR isoform singly or both RyR isoforms in combination, and that utilize CaO-dependent or Ca -independent SR Ca2+ release mechanisms. In the first phase of the project, we will define how each RyR can be activated and the functional properties of the Ca2+ currents and SR Ca2+ release transients associated with each preparation. In the second phase of this work we will investigate the role of Ca2+ release system organization in determining the release process used. Light and electron microscopic techniques will be used to establish the cellular structures and the nonRyR proteins associated with each RyR and each SR Ca2+ release mechanism. In the final phase of this project, we will establish the cellular conditions responsible for the maturation of the Ca2+ release transient. In addition, we will define the properties of the Ca2+ release events mediated by each isoform. Correlations of the results from these studies will permit us to define the functional properties of a two-RyR Ca2+ release system, factors and conditions that determine the release system used, and the contributions made by the different Ca2+ release systems to embryonic muscle development and mature muscle function.